Rapid and label-free immunosensing of Shiga toxin subtypes with surface plasmon resonance imaging

Authors: WANG, BIN - Oak Ridge Institute For Science And Education (ORISE); Park, Bosoon; CHEN, JING - Oak Ridge Institute For Science And Education (ORISE); HE, XIAOHUA - Western Research Institute

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/23/2020
Publication Date: 4/26/2020
Citation: Wang, B., Park, B., Chen, J., He, X. 2020. Rapid and label-free immunosensing of Shiga toxin subtypes with surface plasmon resonance imaging. Toxins. https://doi.org/10.3390/toxins12050280.
DOI: https://doi.org/10.3390/toxins12050280

Interpretive Summary: Shiga toxin is a type of toxic proteins generated from Shiga toxin-producing Escherichia coli (STEC), which is responsible for gastrointestinal diseases reported in numerous outbreaks of foodborne illness around the world. Current methods to detect Shiga toxins have limitation for rapid field-deployable detection with high volume of samples that are needed for regulatory agency. Surface plasmon resonance imaging (SPRi) has proved to be a rapid detection method with advantages including detection of target samples without any sample labelling process, easy-to-use flow cell injection and detection, faster detection time, and simultaneous microarray testing under same conditions. Therefore, SPRi is evaluated in this work for the potential of field-deployable and rapid detection of Shiga toxins. Two toxoids were used as target molecules since they are the less toxic alternatives to Stx1 and Stx2 proteins. Nine antibodies that specifically bind to Shiga toxin 1a and 2a were tested in SPRi to find antibodies with higher capturing capability in this SPRi method. One polyclonal antibody showed the highest sensitivity with short detection time of 20 min. Next, to amplify the SPRi signals from monoclonal antibodies, so-called sandwich immunoassay was formed by gold nanoparticles (GNPs) coated with antibody and the antibodies immobilized on SPRi biochip surface. The sensitivity has been improved to the level of pg/mL. This result proved that the SPRi-based microarray has the potential for rapid detections of different types of Shiga toxins simultaneously.

Technical Abstract: Shiga toxin is responsible for gastrointestinal diseases including the life-threatening hemolytic uremic syndrome (HUS). Rapid detection of Shiga toxin subtypes is critical to monitor the Shiga toxin contamination in food industry. The Shiga toxin alternatives Stx1a and Stx2a toxoids were used in this study to evaluate the SPRi immunoassay for rapid high-throughput detection of Shiga toxins in a flow cell system. Multiple antibodies (Stx1pAb, Stx1-1mAb, Stx1-2mAb, Stx1d-3mAb, Stx1e-4mAb, Stx2pAb, Stx2-1mAb, Stx2-2mAb, and Stx2-10mAb) were spotted on a 50-nm thickness gold film through multiple crosslinking and blocking steps to improve the orientation of antibodies on the biochip surface. Shiga toxins were detected based on the SPRi signal difference ('R) between immobilized testing antibodies and IgG control. Among the antibodies tested, Stx1pAb showed the highest sensitivity for Stx1 toxoid, with the limit of detection (LOD) of 50 ng/mL and detection time of 20 min. Both Stx2-1mAb and Stx2-2mAb exhibited high sensitivity for Stx2 toxoid. Furthermore, gold nanoparticles (GNPs) were used to amplify the SPRi signals of monoclonal antibodies in a sandwich platform. The LOD reached the level of pg/mL with the help of GNP-antibody conjugate. This result proved that SPRi biochip with selected antibodies has the potential for rapid, high-throughput and multiplex detection of Shiga toxins.